Traces of chemical coagulants, such as alum, commonly used in wastewater treatment, remain in the water effluent and also result in secondary sludge that is difficult to treat. On the other hand, Extracellular Polymeric Substances (EPS) are safer and can be used as alternative bio flocculants. A study was hence done to compare the effectiveness of the treatment of Reactive Black 5 (RB5) textile wastewater with chemical coagulants and bacterial EPS. The jar test method was used to treat the wastewater with Alum and Polyferrous Sulphate (PFS) at varied pH (2–10) and respective dose concentrations. EPS was produced by bacteria isolated from cotton gin trash soil collected from Kitui ginnery in Kenya. EPS treatment was done at 30 °C for 72 h. EPS-producing bacteria isolates were identified by molecular analysis. The chemically and EPS-treated wastewater was analyzed for color, Total Dissolved Solids (TDS), and Chemical Oxygen Demand (COD). Respective color and TDS removal capacity by PFS and alum were (86.1% and 42.18%) and (85.1% and 69%) at their optimal conditions of (pH 7 and 100 mg/L) and (pH 7 and 140 mg/L). Similarly, PFS and alum reduced COD by 27.25% and 26.65%. Respective dye removal by YEAK2 −5 and YEPD K2 −2 EPS was 69.1% and 85.7%. However, YEPD K2 −2 and YEAK2 −5 EPS caused a respective rise of TDS by 40.14% and 67.17%. The respective reduction of COD by YEPD K2 −2 and YEAK2 −5 EPS was 56.25% and 54.24%. The chemically and EPS-treated wastewater met the National Environment Management Authority (NEMA) limits. The YEPD K2 −2 and YEAK2 −5 isolates were both identified as Bacillus sp. GC–MS and FTIR analysis results indicated that the two EPS were heteropolysaccharides composed of different sugar derivatives and hydroxyl as the main functional group. The results for treatment of the dye wastewater by Alum, PFS, and EPS were therefore comparable. The bacterial EPS bio flocculants can therefore serve as effective and cleaner substitutes to the alum and PFS chemical coagulants.